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Biomedical subjects

J M Collins

Publications and source records attributed to J M Collins.

At least 181 records · Page 10Linked to original sources
PubMed1979

Electronic sorting of granulocyte precursor cells from stimulated bone marrow.

A commerical cell sorter was used to obtain preparations of cells in various stages of granulocyte development from rabbit marrows stimulated by inflammatory response. Marrow cells were fractionated on density gradients of Ficoll/Hypaque and each fraction sorted using light scatter. Trial and error selection of appropriate gradient fractions and light scatter windows allowed sorting of early (blast cells, promyelocytes), intermediate (myelocytes, metamyelocytes) and late stage (band cells, polys) granulocytes with enhanced purity.

Animals↗
PubMed1978

Rates of DNA synthesis during the S-phase of HeLa cells.

The rates of DNA synthesis were determined for each of two consecutive journeys through S-phase by highly synchronized HeLa cells. Cells at various times after release from the metabolic block were pulsed with [3H]thymidine. The amount of radioactivity in whole cells, purified DNA, and Okazaki fragments provided indexes of the rates of DNA synthesis. Measurements of the average DNA content per cell by the diphenylamine method and the individual DNA content per cell by DNA:propidium fluorescence provided better estimations of the actual rates of DNA synthesis, independent of thymidine metabolism. Unsynchronized cells that had been pulsed with [3H]thymidine were sorted into early, middle, and late S-phase preparations for estimations of the amount of radioactivity per cell. There were differences in the rates predicted by each of the various methods. Rates estimated by fluorescence measurements of DNA content per cell, or by diphenylamine measurements of average DNA content per cell exhibited a pattern of an initial burst, followed by a decreased rate then a final burst. Similar patterns were obtained for the amount of radioactivity in Okazaki fragments, and in early, middle, and late S-phase cells separated from a log-phase culture by electronic cell sorting. Rates estimated by measurements of the amount of radioactivity in whole cells, and the specific activity of purified DNA exhibited a different pattern of an initial slow rate, followed by a maximal rate then a slow rate.

Cell Cycle↗
PubMed1978

RNA synthesis in rat liver cells with different DNA contents.

The DNA content of mature rat liver parenchymal cells falls into three distinct classes with 1:2:4 amounts of DNA. These classes represent 2 N, 4 N, and 8 N cells. We have measured the changing proportions of these cells from 0.75 to 24 months of age. We have obtained a useful approximation for the numbers of each ploidy class, for cellular preparations from liver, as a function of the amount of DNA and age. Multiple linear regression analysis revealed that the average DNA content per cell is given by the relationship DNA/cell = 41.28(1/2)--324.14 Age1/4 + 499.37 Age1/8--207.24, with a multiple correlation coefficient of 0.990. Measurements of the amount of RNA synthesized in a 15-min period revealed that the relative proportions were 1.2:1.0 for 4 N:2 N cells of young rats, 1.3:1.1.:1.0 for 8 N:4 N:2 N cells of intermediate ages, and 1.1:1.0 for 8 N:4 N cells of old rats. The absolute amounts of RNA synthesized by each ploidy class declined with age.

Animals↗
PubMed1978

Isobaric inert gas supersaturation: observations, theory, and predictions.

An isobaric inert gas supersaturation model incorporating both diffusion and perfusion properties of biological tissue is presented in a form which allows ready comparison with experimental observations. This model requires only measurement of inert gas flux and blood gas solubility in order to evaluate "counterdiffusion potential". Inert gas flux across the skin of Yorkshire piglets anesthetized with pentobarbital was measured for He, Ne, CH4, C2H4, N2O, and SF6. Model predictions based upon these data compare favorably with published reports of isobaric inert gas supersaturation, as well as several previously unpublished observations. The possibility of supersaturation resulting from the use of hydrogen as a breathing gas in a helium environment is also discussed, and extensive animal testing is recommended before potentially dangerous human exposure occurs.

Animals↗
PubMed1978

Separation of rabbit marrow precursor cells by combined isopycnic sedimentation and electronic cell sorting.

Separation of developing cells into fractions of differing stages of maturity is critical to effective biochemical study of the process of cellular differentiation. Density gradient techniques utilizing rate-zonal or isopycnic separations have permitted partial separations based on cell mass or cell density. In this study the separation of various rabbit marrow cells was improved by high-speed flow analysis and sorting in a Coulter Two-Parameter Cell Sorter. After preliminary isopycnic separation of marrow cells in Ficoll-Hypaque, cells were sorted into lymphoid and myeloid elements, utilizing light-scatter (LS) profiles to determine sorting. Characteristic LS patterns were present for erythrocytes, lymphocytes, devitalized cells, and granulocytes. When erythrocytes and their precursors were removed by hypotonic lysis, the remaining granulocytes could be sorted to give samples with much greater purity with respect to developmental stage than was possible with density gradients alone. Thus the combination of techniques represents a significant improvement in the ability to study the mechanisms of normal or altered cellular maturation.

Animals↗
PubMed1978

Potential mechanism of macrophage-mediated tumor cell cytotoxicity.

Activated macrophages have been shown to inhibit the incorporation of 125IUDR into the DNA of Lewis lung (LL) tumor cells and to lead to the cytotoxicity of these target cells. Despite this apparent inhibition of DNA synthesis, the increase in the number of LL cells cultured with activated macrophages paralleled that of LL cells alone for the first 18 hours of culture (approximately one doubling of cells). To resolve this paradox, the DNA content of LL cells, which were cultured with activated macrophages for 16 hours, was analyzed by flow microfluorometry. These experiments indicated that subsequent to co-culture with activated macrophages, LL cells with a 50% reduction in their normal DNA content could be detected. Furthermore, after a 2-hour incubation with colcemid, a greater than 95% reduction in metaphase cells occurred in LL cells cultured with activated macrophages. However, dividing nuclei without chromosome condensation appeared to be a prominent feature of these cultures. This would suggest that the genotypic or phenotypic program for cell division in the tumor cells is such that under these unique circumstances of tumor cell-activated macrophage interaction, cell division can proceed in the absence of DNA synthesis. We suggest that this aberrant division may be related to the lethal event which leads to activated macrophage-mediated tumor cell cytotoxicity.

Animals↗
PubMed1978

Improved therapeutic index of bleomycin when administered by continuous infusion in mice.

The effects of three different dosage schedules on both therapeutic effect and pulmonary toxicity of bleomycin were studied in mice. Therapy was assessed by both survival and decreased tumor size in mice bearing Lewis lung carcinoma. Lung toxicity was estimated in nontumored mice as increases in lung collagen content by measuring lung hydroxyproline concentrations. In the first set of experiments, bleomycin injections twice daily (low-dose, high-frequency) produced a significant (34%) increase in lifespan over controls, whereas the same total dose given twice weekly did not result in increased survival. Both schedules produced pulmonary toxicity. Continuous sc infusion of bleomycin via an osmotic minipump was compared to these two schedules of intermittent injection. Identical total doses of drug were administered in all three schedules. Continuous infusion for 7 days produced marked inhibition of tumor growth (T/C = 16%), which was significantly better than twice weekly or ten-times weekly injection of the same total dose. Furthermore, at a total dose of 40 mg/kg of bleomycin, continuous infusion did not result in measurable pulmonary toxicity, whereas both schedules of bolus injection produced significant increases in lung collagen content. Thus, continuous infusion of bleomycin improved its therapeutic effect against Lewis lung carcinoma and also reduced its pulmonary toxicity.

Animals↗
PubMed1977

Structure of parental deoxyribonucleic acid of synchronized HeLa cells.

We have investigated the structure of parental DNA as a function of the cell cycle phase of HeLa cells. DNA was isolated from synchronized HeLa cells 0, 5, 8, and 12 h after release from a second exposure to 2 mM thymidine. These DNA preparations were characterized by CS2SO4/AgClO4 buoyant density, sensitivity to a single-strand specific nuclease, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1 nuclease. The cultures were staged according to cell cycle phase by measurements of DNA content per cell by flow microfluorometry. The cell cycle phases were G1/S (0-h culture), S (5-h culture), G2 (8-h culture), and G1 (12-h culture). There are no nuclease-sensitive sites in G2. As the cells enter G1, the number increases, with a maximum being reached in the S phase. The number of breaks in DNA with respect to cell cycle phase follows the same pattern. The amount of single strandedness, measured by buoyant density and nuclease sensitivity, is also minimal in G2, increases in G1, with a maximum achieved in the S phase. It appears that there is a chromosomal cycle, reflected as continuous structural changes in the DNA molecule, as cells traverse the cell cycle.

Cell Cycle↗
PubMed1977

Deoxyribonucleic acid replication in human diploid fibroblasts stimulated to proliferate.

The elongation of replicating DNA chains was studied in resting (G0) cultures of WI-38 cells stimulated to proliferate. An alkaline sucrose gradient sedimentation method which avoids shear effects was employed. The earliest detected intermediate is a short (4.5 S) piece of DNA which is converted to 11 S and larger (50 to 60 S) pieces within 1 min. The 50 to 60 S pieces are gradually converted to two distinct size classes, 212 S and 275 S, which are in turn converted to a chromosomal aggregate of about 400 S. The overall rate of elongation of DNA is about 1.4 X 10(6) daltons/min.

Cell Line↗
PubMed1977

Deoxyribonucleic acid structure in human diploid fibroblasts stimulated to proliferate.

When contact-inhibited human diploid fibroblasts that have been maintained on medium containing 0.5% serum are stimulated by 10% serum they leave the G0 state and enter the cell cycle at G1. The onset of DNA synthesis occurs at 12 to 13 h. DNA was isolated from cultures that had been stimulated for 0, 2, 4, 6, 12, 16, 20, 24, and 30 h. These DNA preparations were characterized by sensitivity to a single strand specific nuclease, CsSO4/AgClO4 buoyant density, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1-nuclease. The DNA from 4-h stimulated cultures has an increased amount of single strandedness as indicated by sensitivity to S1-nuclease and buoyant density. The amount of single strandedness increases as the cells enter S-phase and decreases as they enter the G2 period. The DNA isolated from G0 cells behaves as native, duplex DNA with virtually no breaks. The DNA from 2-h and 4-h stimulated cultures contained a significant number of breaks. The number of breaks increases to a maximum in S-phase and declines as the cells enter the G2 period. There are no nuclease-sensitive sites (gaps) in G0 or 2-h cultures. The number of sites increases 4 h after stimulation, reaches a maximum during S-phase, and decreases as the cells enter G2. These results lead us to suggest a chromosomal cycle at the level of DNA itself, whereby structural changes are occurring throughout the entire G1 period, aimed toward the initiation of DNA synthesis. These changes reach a maximum degree during replication and decline as replication is terminated.

Cell Division↗